4-1BB polypeptides and DNA encoding 4-1BB polypeptides

ABSTRACT

Novel 4-1BB ligand (4-1BB-L) polypeptides and a human cell surface receptor designated 4-1BB that binds 4-1BB-L are provided. Isolated 4-1BB-L-encoding and human 4-1BB-encoding DNA sequences, recombinant expression vectors comprising the isolated DNA sequences, and host cells transformed with the recombinant expression vectors are disclosed, along with methods for producing the novel polypeptides by cultivating such transformed host cells. Soluble forms of the 4-1BB-L or 4-1BB polypeptides are derived from the extracellular domains thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.08/236,918, filed May 6, 1994, now U.S. Pat. No. 5,674,704, which is acontinuation-in-part of U.S. application Ser. No. 08/060,843, filed May7, 1993, now abandoned.

BACKGROUND OF THE INVENTION

The term “cytokines” encompasses a diverse group of soluble proteinsthat are released by one type of cell and mediate a biological effect onanother cell type. Biological activities exhibited by cytokines includecontrol of proliferation, growth, and differentiation of various celltypes, among which are cells of the hematopoietic or immune systems.

Examples of cytokines include the interleukins (e.g., interleukins 1through 12), the interferons (IFNα, IFNβ, and IFNγ), tumor necrosisfactor (TNFα and TNFβ), epidermal growth factor (EGF), platelet-derivedgrowth factor (PDGF), and colony stimulating factors. Examples of colonystimulating factors (CSF), which control growth and differentiation ofhematopoietic cells, are granulocyte-CSF (G-CSF),granulocyte-macrophage-CSF (GM-CSF), macrophage-CSF (M-CSF or CSF-1),mast cell growth factor (MGF), and erythropoietin (EPO).

The biological activity of cytokines generally is mediated by binding ofthe cytokine to a receptor specific for that cytokine, located on thesurface of target cells. Much research has been directed to identifyingreceptor(s) that bind a given cytokine (often referred to as the“ligand” for the receptor in question), and exploring the roles thatendogenous ligands and receptors play in vivo.

One family of cytokine receptors includes two different TNF receptors(Type I and Type II) (Smith et al., Science 248:1019, 1990) and Schallet al., Cell 61:361, 1990); nerve growth factor receptor (Johnson etal., Cell 47:545, 1986); B cell antigen CD40 (Stamenkovic et al., EMBOJ. 8:1403, 1989); T cell antigen OX40 (Mallett et al., EMBO J. 9:1063,1990); human Fas antigen (Itoh et al., Cell 66:233, 1991); and murinereceptor 4-1BB (Kwon et al., Cell. Immunol. 121:414, 1989) [Kwon et al.I] and Kwon et al., Proc. Natl. Acad. Sci. USA 86:1963, 1989 [Kwon etal. II]).

Expression of murine 4-1BB is induced by concanavalin A (con A) inspleen cells, cloned helper T cells, cytolytic T cells, and cytolytic Tcell hybridomas (Kwon et al. II). Murine 4-1BB cDNA was isolated from acDNA library made from induced RNA isolated from both a helper T cellline and a cytotoxic T cell line (Kwon et al. II). The nucleotidesequence of the isolated cDNA is presented in Kwon et al. II, along withthe amino acid sequence encoded thereby. The murine 4-1BB proteincomprises 256 amino acids, including a putative leader sequence,trans-membrane domain and a number of other features common to cellmembrane bound receptor proteins. Regarding a putative human 4-1BBprotein, neither amino acid nor nucleotide sequence information is knownfor any such protein.

No ligand that would bind 4-1BB and transduce a signal through the 4-1BBreceptor is known. Thus, there is a need in the art to determine whethera novel protein functioning as a ligand for 4-1BB exists, and, if so, toisolate and characterize the 4-1BB ligand protein.

SUMMARY OF THE INVENTION

A novel cytokine designated 4-1BB ligand (4-1BB-L) is disclosed herein.4-1BB-L polypeptides bind to the cell surface receptor designated 4-1BB.Human 4-1BB is also provided by the present invention.

The present invention provides purified 4-1BB-L polypeptides,exemplified by the murine and human 4-1BB-L proteins disclosed herein,and purified human 4-1BB polypeptides. Isolated DNA sequences encoding4-1BB-L or human 4-1BB, recombinant expression vectors comprising theisolated DNA, and host cells transformed with the expression vectors areprovided by the present invention, along with methods for producing4-1BB-L and human 4-1BB by cultivating the transformed host cells.Antibodies that are immunoreactive with 4-1BB-L or human 4-1BB also areprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the results of a competition binding assay thatdemonstrated binding of a murine 4-1BB/Fc fusion protein by a solublemurine 4-1BB-L protein produced in CV-1 (mammalian) cells. The 4-1BB-Lprotein was produced as described in example 7.

FIG. 2 presents the results of the control experiment described inexample 7.

FIG. 3 presents the results of a competition binding assay thatdemonstrated binding of a murine 4-1BB/Fc fusion protein by a solublemurine 4-1BB-L protein produced in yeast cells. The 4-1BB-L protein wasproduced as described in example 8.

FIG. 4 presents the results of an assay described in example 13, inwhich cells expressing recombinant human 4-1BB-L were demonstrated tocostimulate T-cell proliferation. Purified T cells were cultured with atitration of fixed CV-1/EBNA cells that were transfected with eitherempty vector (open circles) or vector containing hu4-1BB-L DNA (closedcircles) in the presence of suboptimal PHA (0.1%). After 3 days,cultures were pulsed with [3H] thymidine and incorporated radioactivitywas assessed 6 hours later. Data are representative of four experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a human cell surface receptor designated4-1BB. Human 4-1BB is a member of the TNF receptor super-family, and isexpressed on cells that include but are not limited to stimulated humanperipheral blood lymphocytes. Expression of murine 4-1BB on cell typesthat include helper, suppressor and cytolytic T lymphocytes has beenreported (Kwon et al. I and II), and 4-1BB has also been detected onmouse brain tissue.

A novel cytokine designated 4-1BB ligand (4-1BB-L) is also providedherein. 4-1BB-L polypeptides bind to the cell surface receptordesignated 4-1BB. Expression of 4-1BB-L has been detected on stimulatedT cells (e.g., the alloreactive CD4⁺ human T cell clone stimulated withanti-CD3 antibodies described in example 5), a subclone of a mousethymoma cell line, mouse brain tissue, and (by RNA analysis) on mousebone marrow, splenic, and thymic tissues.

Purified 4-1BB-L polypeptides, exemplified by the murine and human4-1BB-L proteins disclosed herein, and purified human 4-1BB polypeptidesare encompassed by the present invention. Isolated DNA sequencesencoding 4-1BB-L or human 4-1BB, recombinant expression vectorscomprising the isolated DNA, and host cells transformed with theexpression vectors are provided by the present invention, along withmethods for producing 4-1BB-L and human 4-1BB by cultivating thetransformed host cells and purifying the recombinant protein. Antibodiesthat are immunoreactive with 4-1BB-L or human 4-1BB are also disclosed.

The present invention provides full length 4-1BB-L and 4-1BBpolypeptides as well as biologically active fragments and variantsthereof. Soluble polypeptides comprising the extracellular domain of4-1BB-L or a receptor-binding fragment thereof are among thebiologically active fragments provided. Likewise, soluble polypeptidesderived from the extracellular domain of human 4-1BB that are capable ofbinding the 4-1BB ligand are encompassed by the present invention. Suchsoluble polypeptides are described in more detail below.

4-1BB-L refers to a genus of mammalian polypeptides that are capable ofbinding 4-1BB. 4-1BB-L is a type II extracellular membrane polypeptidewith an intracellular (cytoplasmic) domain at the N-terminus of thepolypeptide, followed by a transmembrane region, and an extracellular(receptor-binding) domain at the C-terminus of the polypeptide. Soluble4-1BB-L polypeptides may be derived from the extracellular domain, asdescribed below. While not wishing to be bound by theory, binding of the4-1BB ligand to 4-1BB may initiate transduction of a biological signalin a cell bearing the receptor.

cDNA encoding murine 4-1BB-L was isolated using a direct expressioncloning technique, as described in example 4. Briefly, a fusion proteincomprising a fragment of the murine 4-1BB extracellular (ligand-binding)domain fused to the Fc domain of a human IgG1 antibody was prepared andused to screen an expression cloning cDNA library derived from asubclone of a mouse thymoma cell line. A clone expressing a protein thatbound the 4-1BB/Fc fusion protein was identified, sequenced anddetermined to encode a novel protein, which is a ligand for 4-1BB. Thenucleotide sequence of the murine 4-1BB-L cDNA isolated by thisprocedure and the amino acid sequence encoded thereby are presented inSEQ ID NO:1 and SEQ ID NO:2. This murine 4-1BB-L protein comprises acytoplasmic domain (amino acids 1–82 of SEQ ID NO:2), a transmembraneregion (amino acids 83–103), and an extracellular domain (amino acids104–309).

A direct expression cloning technique also was used to isolate cDNAencoding a human 4-1BB-L, as described in example 5. Briefly, anexpression cloning cDNA library derived from an alloreactive CD4⁺ humanT cell clone stimulated with an anti-CD3 antibody was screened with afusion protein comprising a soluble human 4-1BB polypeptide fused to anFc polypeptide. The nucleotide sequence of a human 4-1BB-L cDNA isolatedby this procedure and the amino acid sequence encoded thereby arepresented in SEQ ID NO:3 and SEQ ID NO:4. This human 4-1BB-L proteincomprises a cytoplasmic domain (amino acids 1–25 of SEQ ID NO:4), atransmembrane region (amino acids 26–48), and an extracellular domain(amino acids 49–254).

The nucleotide sequence of a human 4-1BB cDNA (isolated as described inexample 2) and the amino acid sequence encoded thereby are presented inSEQ ID NO:7 and SEQ ID NO:8. The human 4-1BB protein comprises anN-terminal signal sequence (amino acids −23 to −1 of SEQ ID NO:8), anextracellular domain comprising amino acids 1–163, a transmembraneregion comprising amino acids 164–190, and a cytoplasmic domaincomprising amino acids 191–232. The human 4-1BB amino acid sequence ofSEQ ID NO:8 is 60% identical to that of the murine 4-1BB receptordescribed in Kwon et al. (Proc. Natl. Acad. Sci. USA 86:1963, 1989).

Also encompassed by the present invention are isolated DNA sequencesthat are degenerate as a result of the genetic code to the nucleotidesequence of SEQ ID NOS:1, 3, or 7 (and thus encode the amino acidsequence presented in SEQ ID NOS: 2, 4, or 8). The 4-1BB-L nucleotidesequences of SEQ ID NOS:1, 3, or 7 are understood to include thesequences complementary thereto.

Purified human 4-1BB-L proteins characterized by the N-terminal aminoacid sequence Met-Glu-Tyr-Ala-Ser-Asp-Ala-Ser-Leu-Asp-Pro-Glu- or(beginning with the first amino acid of the extracellular domain)Leu-Ala-Cys-Pro-Trp-Ala-Val-Ser-Gly-Ala-Arg-Ala-Ser- are providedherein. Purified murine 4-1BB-L proteins characterized by an N-terminalamino acid sequence selected from the group consisting ofMet-Asp-Gln-His-Thr-Leu-Asp-Val-Glu-Asp-Thr-Ala-, or (beginning with oneof the first three amino acids of the extracellular domain)Arg-Thr-Glu-Pro-Arg-Pro-Ala-Leu-Thr-Ile-Thr-Thr-,Thr-Glu-Pro-Arg-Pro-Ala-Leu-Thr-Ile-Thr-Thr-, andGlu-Pro-Arg-Pro-Ala-Leu-Thr-Ile-Thr-Thr- are also disclosed herein.

Soluble Proteins and Multimeric Forms of the Inventive Proteins

Soluble forms of the 4-1BB-L and 4-1BB proteins are provided herein.Soluble 4-1BB-L or 4-1BB polypeptides comprise all or part of theextracellular domain but lack the transmembrane region that would causeretention of the polypeptide on a cell membrane. The solublepolypeptides that may be employed retain the ability to bind 4-1BB. Thesoluble 4-1BB polypeptides that may be employed retain the ability tobind the 4-1BB ligand. The soluble proteins may include part of thetransmembrane region or part of the cytoplasmic domain, provided thatthe protein is capable of being secreted rather than retained on thecell surface.

Since the 4-1BB-L protein lacks a signal peptide, a heterologous signalpeptide advantageously is fused to the N-terminus of soluble 4-1BB-Lpolypeptides to promote secretion thereof. The signal peptide is cleavedfrom the protein upon secretion from the host cell. The need to lyse thecells and recover the recombinant soluble protein from the cytoplasmthus is avoided. The native signal peptide or a heterologous signalpeptide (such as one of the heterologous signal peptides describedbelow, chosen according to the intended expression system)advantageously is fused to a soluble 4-1BB polypeptide.

Soluble proteins of the present invention may be identified (anddistinguished from their non-soluble membrane-bound counterparts) byseparating intact cells expressing the desired protein from the culturemedium, e.g., by centrifugation, and assaying the medium (supernatant)for the presence of the desired protein. The culture medium may beassayed using procedures which are similar or identical to thosedescribed in the examples below.

Soluble forms of the 4-1BB-L and 4-1BB proteins are advantageous forcertain applications, e.g., when the protein is to be administeredintravenously for certain therapeutic purposes. Also, purification ofthe proteins from recombinant host cells is facilitated, since thesoluble proteins are secreted from the cells. In one embodiment of theinvention, a soluble fusion protein comprises a first polypeptidederived from the extracellular domain of 4-1BB or 4-1BB-L fused to asecond polypeptide added for purposes such as facilitating purificationor effecting dimer formation. Suitable second polypeptides do notinhibit secretion of the soluble fusion protein.

Examples of soluble polypeptides include those comprising the entireextracellular domain. Representative examples of the soluble proteins ofthe present invention include, but are not limited to, a polypeptidecomprising amino acids x-309 of SEQ ID NO:2, wherein x is selected from104, 105, and 106 (murine 4-1BB-L); amino acids 49–254 of SEQ ID NO:4(human 4-1BB-L); or amino acids 1–163 of SEQ ID NO:8 (human 4-1BB).Preparation of certain soluble polypeptides of the present invention isdescribed in the examples section.

Truncated forms of the inventive proteins, including solublepolypeptides, may be prepared by any of a number of conventionaltechniques. In the case of recombinant proteins, a DNA fragment encodinga desired fragment may be subcloned into an expression vector.Alternatively, a desired DNA sequence may be chemically synthesizedusing known techniques. DNA fragments also may be produced byrestriction endonuclease digestion of a full length cloned DNA sequence,and isolated by electrophoresis on agarose gels. Linkers containingrestriction endonuclease cleavage site(s) may be employed to insert thedesired DNA fragment into an expression vector, or the fragment may bedigested at cleavage sites naturally present therein. The well knownpolymerase chain reaction procedure also may be employed to isolate aDNA sequence encoding a desired protein fragment by usingoligonucleotide primers comprising sequences that define the termini ofthe desired fragment.

In another approach, enzymatic treatment (e.g., using Bal 31exonuclease) may be employed to delete terminal nucleotides from a DNAfragment to obtain a fragment having a particular desired terminus.Among the commercially available linkers are those that can be ligatedto the blunt ends produced by Bal 31 digestion, and which containrestriction endonuclease cleavage site(s). Alternatively,oligonucleotides that reconstruct the N- or C-terminus of a DNA fragmentto a desired point may be synthesized. The oligonucleotide may contain arestriction endonuclease cleavage site upstream of the desired codingsequence and position an initiation codon (ATG) at the N-terminus of thecoding sequence. present therein. The well known polymerase chainreaction procedure also may be employed to isolate a DNA sequenceencoding a desired protein fragment by using oligonucleotide primerscomprising sequences that define the termini of the desired fragment.

Naturally occurring soluble forms of 4-1BB-L or human 4-1BB are alsoencompassed by the present invention. Such soluble polypeptides mayresult from alternative splicing of mRNA during expression, or releaseof a soluble polypeptide from a membrane-bound form of the protein byproteolysis.

Oligomeric (multimeric) forms of the inventive proteins are encompassedby the present invention. The terms “inventive proteins” and “inventivepolypeptides” as used herein refer collectively to the 4-1BB-L and 4-1BBproteins or polypeptides of the present invention, as defined by theappended claims. The oligomers preferably are dimers or trimers. Dimericand trimeric forms of the 4-1BB-L and 4-1BB proteins may exhibitenhanced biological activity compared to the monomeric forms. Separatepolypeptide chains may be joined by interchain disulfide bonds formedbetween cysteine residues to form oligomers. Alternatively, themultimers may be expressed as fusion proteins, with or without spaceramino acids between the inventive protein moieties, using recombinantDNA techniques. In one embodiment of the present invention, two or threesoluble 4-1BB-L or 4-1BB polypeptides are joined via a polypeptidelinker (e.g., one of the antibody-derived or peptide linkers describedbelow).

In one embodiment of the present invention, a soluble fusion proteincomprises a soluble 4-1BB or 4-1BB-L polypeptide fused to a polypeptidederived from the constant region of an antibody. Multimers resultingfrom formation of interchain disulfide bonds between theantibody-derived moieties of such fusion proteins are provided.

Examples of such fusion proteins are those comprising one of theabove-described soluble 4-1BB or 4-1BB-L polypeptides fused to anantibody Fc region polypeptide. A gene fusion encoding the fusionprotein is inserted into an appropriate expression vector and cellstransformed with the expression vector are cultured to produce andsecrete the fusion protein. The expressed fusion proteins are allowed toassemble much like antibody molecules, whereupon interchain disulfidebonds form between Fc polypeptides, yielding the Fc/4-1BB-L or 4-1BB/Fcprotein in dimeric form. The preparation of certain embodiments of suchfusion proteins and dimers formed therefrom is described in more detailin the examples section below. If two different fusion proteins aremade, one comprising an inventive protein fused to the heavy chain of anantibody and the other comprising an inventive protein fused to thelight chain of an antibody, it is possible to form oligomers comprisingas many as four soluble inventive polypeptides.

Preparation of fusion proteins comprising heterologous polypeptidesfused to various portions of antibody-derived polypeptides (includingthe Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA88:10535, 1991) and Byrn et al. (Nature 344:677, 1990). The term “Fcpolypeptide” includes native and mutein forms, as well as truncated Fcpolypeptides containing the hinge region that promotes dimerization. Oneexample is an Fc region encoded by cDNA obtained by PCR as described byFanslow et al., J. Immunol. 149:65 (1992). One example of a DNA encodinga mutein of the Fc region of a human IgG1 antibody is described in U.S.patent application Ser. No. 08/097,827, entitled “Novel Cytokine Whichis a Ligand for OX40” filed Jul. 23, 1993, which application is herebyincorporated by reference. The mutein DNA was derived from a native Fcpolypeptide-encoding DNA by site-directed mutagenesis conductedessentially as described by Deng and Nickoloff, Anal. Biochem. 200:81(1992). The amino acid sequence of the Fc mutein polypeptide isidentical to that of the native Fc polypeptide presented in SEQ ID NO:15except that amino acid 32 of SEQ ID NO:15 has been changed from Leu toAla, amino acid 33 has been changed from Leu to Glu, and amino acid 35has been changed from Gly to Ala. This mutein Fc exhibits reducedaffinity for immunoglobulin receptors.

Alternatively, one can link multiple copies of the inventive proteinsvia peptide linkers. A fusion protein comprising two or more copies ofthe inventive protein, separated by peptide linkers, may be produced byrecombinant DNA technology. Among the peptide linkers that may beemployed are amino acid chains that are from 5 to 100 amino acids inlength, preferably comprising amino acids selected from the groupconsisting of glycine, asparagine, serine, threonine, and alanine. Inone embodiment of the present invention, a fusion protein comprises twoor three soluble 4-1BB-L or 4-1BB polypeptides linked via a peptidelinker selected from Gly₄SerGly₅Ser (SEQ ID NO: 16) and (Gly₄Ser)_(n)(SEQ ID NO: 17), wherein n is 4–12. The production of recombinant fusionproteins comprising peptide linkers is illustrated in U.S. Pat. No.5,073,627, for example.

The 4-1BB-L proteins of the present invention are believed to be capableof dimerization without having one of the above-describedantibody-derived polypeptides fused to the ligand. Both soluble and fulllength recombinant 4-1BB-L proteins have been precipitated with 4-1BB/Fc(reductive immunoprecipitation) followed by purification by affinitychromatography on a column containing protein G. Dimers were detected bySDS-PAGE (non-reducing gel). Higher oligomers may have formed as well.Thus, fusing polypeptides that promote dimerization (or formation ofhigher oligomers) to 4-1BB ligands may result in undesirable aggregateformation.

Variants and Derivatives

As used herein, the terms “4-1BB-L” and “human 4-1BB” include variantsand derivatives that retain a desired biological activity of the nativemammalian polypeptides. The variant sequences differ from a nativenucleotide or amino acid sequence by one or a plurality ofsubstitutions, deletions, or additions, but retain a desired biologicalactivity such as the ability to bind 4-1BB (for variants of 4-1BB-L) orthe ability to bind a 4-1BB-L (for variants of 4-1BB, the receptor).Derivatives of the inventive proteins may comprise moieties such as thechemical moieties described below, attached to the inventive protein.

In one embodiment of the present invention, a variant sequence issubstantially identical to a native sequence. The term “substantiallyidentical” as used herein means that the amino acid or nucleotidesequence in question is at least 80% identical, preferably 90–100%identical, to a reference (native) sequence. The degree of homology(percent identity) may be determined, for example, by comparing sequenceinformation using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The GAP programutilizes the alignment method of Needleman and Wunsch (J. Mol. Biol.48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482,1981). The preferred default parameters for the GAP program include: (1)a unary comparison matrix (containing a value of 1 for identities and 0for non-identities) for nucleotides, and the weighted comparison matrixof Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described bySchwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure,National Biomedical Research Foundation, pp. 353–358, 1979; (2) apenalty of 3.0 for each gap and an additional 0.10 penalty for eachsymbol in each gap; and (3) no penalty for end gaps.

Alterations of the native amino acid sequence may be accomplished by anyof a number of known techniques, e.g., by mutation of the nativenucleotide sequences disclosed herein. Mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analog having the desired amino acid insertion,substitution, or deletion. Alternatively, oligonucleotide-directedsite-specific mutagenesis procedures such as those described by Walderet al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik(BioTechniques, January 1985, 12–19); Smith et al. (Genetic Engineering:Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos.4,518,584 and 4,737,462, may be employed.

Isolated DNA sequences that hybridize to the murine 4-1BB-L-encodingnucleotide sequence of SEQ ID NO:1 or the human 4-1BB-L-encodingnucleotide sequence of SEQ ID NO:3 under moderately stringent orseverely stringent conditions are encompassed by the present invention.Moderate stringency conditions refer to conditions described in, forexample, Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed.Vol. 1, pp. 1.101–104, Cold Spring Harbor Laboratory Press, (1989).Conditions of moderate stringency, as defined by Sambrook et al.,include prewashing in 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) andhybridization at about 55° C. in 5×SSC overnight. Conditions of severestringency include higher temperatures of hybridization and washing. Theskilled artisan recognizes that the temperature and wash solution saltconcentration may be adjusted as necessary according to factors such asthe length of the probe. One embodiment of the invention is directed toDNA sequences that will hybridize under severely stringent conditions toa DNA sequence comprising the coding region of a 4-1BB-L clone disclosedherein. The severely stringent conditions include hybridization at 68°C. followed by washing in 0.1×SSC/0.1% SDS at 63–68° C.

Among the hybridizing sequences encompassed by the present invention arethose encoding a biologically active primate or murine 4-1BB-Lpolypeptides. Biologically active polypeptides encoded by DNA sequencesthat hybridize to the murine 4-1BB-L-encoding nucleotide sequence of SEQID NO:1 or the human 4-1BB-L-encoding nucleotide sequence of SEQ ID NO:3under moderately stringent or severely stringent conditions areencompassed by the present invention.

In one embodiment of the present invention, a variant amino acidsequence comprises conservative amino acid substitution(s) but isotherwise identical to a native amino acid sequence. Conservativesubstitutions refer to replacement of a given amino acid residue with aresidue having similar physiochemical characteristics. Examples ofconservative substitutions include substitution of one aliphatic residuefor another, such as Ile, Val, Leu, or Ala for one another, orsubstitutions of one polar residue for another, such as between Lys andArg; Glu and Asp; or Gln and Asn. Other such conservative substitutions,for example, substitutions of entire regions having similarhydrophobicity characteristics, are well known.

The present invention further includes the inventive polypeptides withor without associated native-pattern glycosylation. The recombinantproteins when expressed in yeast or mammalian expression systems (e.g.,COS-7 cells) may be similar or significantly different in molecularweight and glycosylation pattern than the corresponding native proteins.Expression of mammalian 4-1BB-L polypeptides in bacterial expressionsystems such as E. coli, provides non-glycosylated molecules

Variant proteins comprising inactivated N-glycosylation sites are withinthe scope of the present invention. Such variants are expressed in amore homogeneous, reduced carbohydrate form. N-glycosylation sites ineukaryotic polypeptides are characterized by an amino acid tripletAsn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Inthis sequence, carbohydrate residues are covalently attached at the Asnside chain. Addition, substitution, or deletion of residue(s) so thatthe Asn-X-Y triplet is no longer present inactivates the site. In oneembodiment, a conservative amino acid substitution replaces the Asnresidue, with substitution of Asp, Gln, or Glu for Asn being preferred.Known procedures for inactivating N-glycosylation sites in proteinsinclude those described in U.S. Pat. No. 5,071,972 and EP 276,846.

The murine 4-1BB-L of SEQ ID NO:2 comprises three N-glycosylation sites,at residues 139–141, 161–163, and 293–295. The human 4-1BB-L of SEQ IDNO:4 comprises no N-glycosylation sites. The human 4-1BB of SEQ ID NO:8comprises two such sites, at residues 115–117 and 126–128.

Naturally occurring variants such as those resulting from alternativemRNA splicing events or proteolytic cleavage are also within the scopeof the present invention. Variations attributable to proteolysisinclude, for example, differences in the N- or C-termini upon expressionin different types of host cells, due to proteolytic removal of one ormore terminal amino acids (which may occur intracellularly or duringpurification). In one embodiment of the present invention, the inventiveproteins lack from one to five of the N- or C-terminal amino acids ofthe sequences disclosed herein. In certain host cells,post-translational processing will remove the methionine residue encodedby an initiation codon, whereas the methionine residue will remain atthe N-terminus of proteins produced in other host cells.

Additional variants may be prepared by deleting terminal or internalsequences not needed for biological activity. For example, Cys residuescan be deleted or replaced with other amino acids to prevent formationof incorrect intramolecular disulfide bridges upon renaturation.

Other variants are prepared by modifying KEX2 protease processing sitesin the inventive proteins to enhance expression in yeast cells in whichKEX2 protease activity is present. The adjacent basic residue pairs thatconstitute KEX2 protease processing sites, and are to be inactivated byadding, substituting or deleting residue(s), are Arg-Arg, Arg-Lys, andLys-Arg pairs. Lys-Lys pairs are considerably less susceptible to KEX2cleavage, and conversion of Arg-Arg, Arg-Lys, and Lys-Arg pairs to aLys-Lys doublet is a conservative and preferred alteration thatessentially inactivates the KEX2 sites. EP 1212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein.

The inventive proteins may be modified by forming covalent oraggregative conjugates with other chemical moieties, such as glycosylgroups, lipids, phosphate, acetyl groups and the like. Covalentderivatives are prepared by reaction of functional groups of thechemical moiety with functional groups on amino acid side chains or atthe N-terminus or C-terminus of the inventive protein. Also providedherein are the inventive proteins comprising detectable labels,diagnostic or cytotoxic reagents attached thereto, including but notlimited to radionuclides, colorimetric reagents, and the like.

Other derivatives within the scope of this invention include covalent oraggregative conjugates of the inventive proteins or fragments thereofwith other proteins or polypeptides, such as by synthesis in recombinantculture as N-terminal or C-terminal fusions. The inventive proteins cancomprise polypeptides added to facilitate purification andidentification (e.g., the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and Hopp et al., Bio/Technology 6:1204, 1988; ora poly-His peptide). One such peptide is the FLAG® peptide D, which is ahighly antigenic sequence that provides an epitope reversibly bound by aspecific monoclonal antibody (e.g., the monoclonal antibody produced bythe hybridoma designated 4E11 and deposited with the American TypeCulture Collection under accession no. HB 9259) to enable rapid assayand facilitate purification of the expressed recombinant polypeptidefused thereto.

Assays for Biological Activity

The 4-1BB-L and 4-1BB proteins of the present invention and variants andderivatives thereof may be tested for biological activity by anysuitable assay procedure. The procedure will vary according to suchfactors as whether the protein to be tested is bound to a cell surfaceor is secreted into the culture supernatant. Proteins may beradiolabeled for use in the assays, e.g., using the commerciallyavailable IODO-GEN reagent described in example 1.

Competitive binding assays can be performed using standard methodology.For example, a 4-1BB-L variant can be tested for the ability to competewith a radiolabeled 4-1BB-L protein for binding to cells that express4-1BB on the cell surface. Likewise, a 4-1BB variant can be assayed forthe ability to compete with a radiolabeled 4-1BB for binding to cellsexpressing membrane-bound 4-1BB-L. Qualitative results can be obtainedby competitive autoradiographic plate binding assays, or Scatchard plotsmay be utilized to generate quantitative results. Instead of intactcells, one could substitute a 4-1BB or 4-1BB-L protein bound to a solidphase such as a column chromatography matrix (e.g. a soluble 4-1BB/Fcfusion protein bound to a Protein A or Protein G column by interactionwith the Fc region of the fusion protein).

Intact cells employed in competition binding assays may be cells thatnaturally express 4-1BB-L or 4-1BB (e.g., cell types identified in theexamples below). Alternatively, cells transfected with recombinantexpression vectors such that the cells express 4-1BB-L or 4-1BB.

One assay technique useful for intact cells expressing a membrane-boundform of the protein in question is the phthalate oil separation method(Dower et al. J. Immunol. 132:751 (1984)), essentially as described byPark et al. (J. Biol. Chem. 261:4177 (1986)). Sodium azide (0.2%) can beincluded in a binding assay to inhibit internalization of 4-1BB-L by thecells. Cells expressing 4-1BB on their surface can be tested forradiolabeled 4-1BB-L binding by a plate binding assay as described inSims et al., Science 241:585 (1988).

Expression Systems

The present invention provides recombinant expression vectors forexpression of the proteins of the present invention and host cellstransformed with the expression vectors. Any suitable expression systemmay be employed.

Recombinant expression vectors of the present invention comprise DNAencoding a 4-1BB-L polypeptide or a human 4-1BB polypeptide, operablylinked to regulatory sequence(s) suitable for expression of said DNAsequence in a host cell. The 4-1BB-L or 4-1BB-encoding DNA may comprisecDNA, genomic DNA, chemically synthesized DNA, DNA isolated by PCR, orcombinations thereof. The regulatory sequences may be derived fromsources that include, but are not limited to, mammalian, microbial,viral, or insect genes. Examples of regulatory sequences includepromoters, operators, and enhancers, ribosomal binding sites, andappropriate sequences that control transcription and translationinitiation and termination. Nucleotide sequences are operably linkedwhen the regulatory sequence functionally relates to the structuralgene. For example, a promoter sequence is operably linked to a codingsequence (e.g. structural gene DNA) if the promoter controls thetranscription of the structural gene.

Suitable host cells for expression of the inventive proteins includeprokaryotes, yeast or higher eukaryotic cells, with mammalian cellsbeing preferred. The recombinant expression vectors are transfected intothe host cells by conventional techniques. The transfected cells arecultivated under conditions suitable to effect expression of the desiredrecombinant protein, which is purified from the cells or culture medium,depending on the nature of the culture system and the expressed protein.As will be readily appreciated by the skilled artisan, cultivationconditions will vary according to factors that include the type of hostcell and particular expression vector employed. Cell-free in vitrotranslation systems could also be employed to produce the inventiveproteins by translation of mRNA complementary to a nucleotide sequencedisclosed herein.

Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described, for example,in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, N.Y.,(1985). Expression vectors generally comprise one or more phenotypicselectable markers (e.g., a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement) andan origin of replication recognized by the intended host cell to ensureamplification within the host.

Certain prokaryotic expression vectors may be constructed by inserting apromoter and other desired regulatory sequences into a commerciallyavailable plasmid such as the cloning vector pBR322 (ATCC 37017). pBR322contains genes for ampicillin and tetracycline resistance and thusprovides simple means for identifying transformed cells. Promoterscommonly employed in prokaryotic expression vectors include β-lactamase(penicillinase), the lactose promoter system (Chang et al., Nature275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan(trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980;and EP-A-36,776) and tac promoter (Maniatis, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). Aparticularly useful prokaryotic host cell expression system employs aphage λP_(L) promoter and a cI857ts thermolabile repressor sequence.Plasmid vectors available from the American Type Culture Collectionwhich incorporate derivatives of the λP_(L) promoter include plasmidpHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) and pPLc28(resident in E. coli RR1 (ATCC 53082)).

The inventive proteins may be expressed in yeast host cells, preferablyfrom the Saccharomyces genus (e.g., S. cerevisiae). Other genera ofyeast, such as Pichia or Kluyveromyces, may also be employed. Yeastvectors commonly contain an origin of replication from a 2μ yeastplasmid, an autonomously replicating sequence (ARS), a promoter region,sequences for polyadenylation, sequences for transcription termination,and a selectable marker. Suitable promoter sequences for yeast vectorsinclude promoters for metallothionein, 3-phosphoglycerate kinase(Hitzeman et al., J. Biol. Chem. 255:2073 (1980) or other glycolyticenzymes (Hess et al., J. Adv. Enzyme Reg. 7:149 (1968) and Holland etal., Biochem. 17:4900 (1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. The ADH2 promoter has beendescribed by Russell et al. (J. Biol. Chem. 258:2674 (1982)) and Beieret al. (Nature 300:724 (1982)). Other suitable vectors and promoters foruse in yeast expression are further described in Hitzeman, EPA-73,657.The vector may comprise a sequence encoding the yeast α-factor leader todirect secretion of a heterologous protein (an inventive protein) fusedthereto. See Kurjan et al., Cell 30:933, 1982; and Bitter et al., Proc.Natl. Acad. Sci. USA 81:5330, 1984.

Shuttle vectors replicable in more than one type of cell comprisemultiple origins of replication and selective markers. For example, ashuttle vector that replicates in both yeast and E. coli and functionsas an expression vector in yeast may comprise DNA sequences from pBR322for selection and replication in E. coli (Amp^(r) gene and origin ofreplication) and yeast-derived sequences such as a glucose-repressibleADH2 promoter, an origin of replication from a 2μ yeast plasmid, and anα-factor leader sequence.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929 (1978). The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or insect host cell culture systems could also be employed toexpress the recombinant proteins of the present invention. Examples ofsuitable mammalian host cell lines include the COS-7 lines of monkeykidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175 (1981)), Lcells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO)cells, HeLa cells, CV-1 cells, CV-1/EBNA cells and BHK (ATCC CRL 10)cell lines. Suitable mammalian expression vectors generally includenontranscribed elements such as an origin of replication, a promotersequence, an enhancer linked to the structural gene, other 5′ or 3′flanking nontranscribed sequences, such as ribosome binding sites, apolyadenylation site, splice donor and acceptor sites, andtranscriptional termination sequences.

Transcriptional and translational control sequences in mammalian hostcell expression vectors may be provided by viral sources. For example,commonly used mammalian cell promoter sequences and enhancer sequencesare derived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), andhuman cytomegalovirus. DNA sequences derived from the SV40 viral genome,for example, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a structural gene sequence in a mammalianhost cell. Viral early and late promoters are particularly usefulbecause both are easily obtained from a viral genome as a fragment whichmay also contain a viral origin of replication (Fiers et al., Nature273:113 (1978)). Smaller or larger SV40 fragments may also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

Exemplary mammalian expression vectors can be constructed as disclosedby Okayama and Berg (Mol. Cell. Biol. 3:280 (1983)). A useful highexpression vector, PMLSV N1/N4, described by Cosman et al., Nature312:768 (1984) has been deposited as ATCC 39890. A vector designatedpHAVEO is described by Dower et al., J. Immunol. 142:4314 (1989).Certain useful mammalian expression vectors are described in theexamples section below.

The vectors additionally may contain a DNA sequence encoding a signalpeptide (secretory leader) fused to the 5′ end of a DNA sequenceencoding one of the inventive polypeptides. The 4-1BB-L polypeptideslack a native signal sequence. Replacement of the native human 4-1BBsignal sequence with a heterologous signal sequence may be desirable toenhance expression levels in the particular host cells employed.Examples of heterologous signal peptides that may be employed are thehuman or murine interleukin-7 signal peptide described in U.S. Pat. No.4,965,195; the interleukin-2 signal peptide described in Cosman et al.Nature 312:768, 1984; and the interleukin-4 signal peptide described inEP 367,566.

Purification of Recombinant Mammalian 4-1BB-L

The present invention provides substantially homogeneous 4-1BB-L andhuman 4-1BB proteins, which may be produced by recombinant expressionsystems or purified from naturally occurring cellular sources. Theproteins are purified to substantial homogeneity, as indicated by asingle protein band upon analysis by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE).

Recombinant 4-1BB or 4-1BB-L proteins may be produced as follows. Hostcells are transformed with an expression vector containing DNA encodingan inventive polypeptide, wherein the DNA is operably linked toregulatory sequences suitable for effecting expression of said inventivepolypeptide in the particular host cells. The transformed host cells arecultured under conditions that promote expression of the 4-1BB-L or4-1BB polypeptide, which is then purified from the culture media or cellextracts. The purification procedure will vary according to such factorsas the particular host cells employed and whether the expressed proteinis secreted or membrane-bound, as the skilled artisan will readilyappreciate.

Recombinant protein produced in bacterial culture is usually isolated byinitial disruption of the host cells, centrifugation, extraction fromcell pellets if the desired protein is in the form of an insolublerefractile body, or from the supernatant if a soluble polypeptide,followed by one or more concentration, salting-out, ion exchange or sizeexclusion chromatography steps. Finally, RP-HPLC can be employed forfinal purification steps. Microbial cells can be disrupted by anyconvenient method, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Recombinant polypeptides secreted from yeast cells can be purified bymethods analogous to those disclosed by Urdal et al. (J. Chromatog.296:171 (1984)). Urdal et al. describe two sequential, reversed-phaseHPLC steps for purification of recombinant human IL-2 on a preparativeHPLC column.

The purification procedure may involve affinity chromatography. A4-1BB-L protein (or the extracellular domain thereof) may be attached toa solid support material by standard procedures for use in purifying a4-1BB protein. Likewise, a 4-1BB protein (or the extracellular domainthereof) attached to a solid support material may be used in purifying a4-1BB-L protein. In addition, 4-1BB-L/Fc or 4-1BB/Fc fusion proteins maybe attached to Protein G- or Protein A-bearing chromatography columnsvia binding of the Fc moiety to the Protein A or Protein G.Immunoaffinity columns comprising an antibody that binds the desiredinventive protein (described in example 8) also may be employed.

In one purification procedure, a 4-1BB-L or 4-1BB polypeptide isconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Alternatively, a cation exchange stepcan be employed. Suitable cation exchangers include various insolublematrices comprising sulfopropyl or carboxymethyl groups. Sulfopropylgroups are preferred.

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify 4-1BB-L. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous recombinant protein.

Pharmaceutical Compositions Comprising the Inventive Polypeptides andUses of 4-1BB-L and 4-1BB DNA and Proteins

The 4-1BB and 4-1BB-L proteins of the present invention are expressed oncells that include certain types of T-lymphocytes, as discussed aboveand in the examples section. The inventive proteins thus are useful inexploring mechanisms of T-cell activation. Identifying novel proteinsexpressed on T-cells, such as the inventive proteins disclosed herein,has important implications in furthering understanding of the regulationand function of the immune system.

Murine 4-1BB and 4-1BB-L also have been detected on brain tissue.Northern blot analysis revealed expression of human 4-1BB-L in brain,and human 4-1BB is expected to be expressed in the brain as well. Theinventive proteins are useful reagents for studying neural tissue, e.g.,research into growth of neural cells and disorders of the brain.

The 4-1BB-L of the present invention also has been found to stimulategrowth of CD3⁻ CD4⁻ CD8⁻ immature lymphocytes. Cells expressing amembrane-bound 4-1BB-L were cultivated with CD3⁻ CD4⁻ CD8⁻ immaturelymphocytes, and growth of the lymphocytes was stimulated.

As described in example 13, cells expressing recombinant human 4-1BB-Linduced a strong proliferative response in mitogen costimulatedperipheral blood T-cells. In contrast, the ligand enhanced cytolysisseen in costimulated long-term cultured T-cell clones.

Uses of 4-1BB-L that flow from this ligand's ability to co-stimulateT-cell proliferation include, but are not limited to, the following.4-1BB-L finds use as a tissue culture reagent for the in vitrocultivation of primary T-cells, and during the derivation of clonalT-cell lines therefrom. The ligand also may be employed to stimulateproliferation of activated T-cells that are to be employed intherapeutic procedures. For example, T-cells may be removed from acancer patient and cultivated in the presence of a tumor antigen invitro by known procedures, to generate cytotoxic T-lymphocytes (CTLs)specific for the patient's tumor cells. The CTLs are then administeredto the patient. To enhance proliferation of the CTLs in the ex vivostage, 4-1BB-L may be added to the culture medium, either alone or incombination with other cytokines such as interleukin-2.

It has been suggested that elimination of peripheral T-cells byactivation induced cytolysis may be an important mechanism of regulatingunwanted or autoreactive T-cells (Owen-Schaub et al., Cell. Immunol.140:197, 1992). 4-1BB-L enhanced cell death induced by mitogenic stimuliin a long-term cultured (chronically activated) T-cell clone. These datasuggest that 4-1BB-L may play a role in this process by enhancingactivation-induced cell death.

The 4-1BB-L of the present invention is useful as a research reagent inin vitro binding assays to detect cells expressing 4-1BB. For example,4-1BB-L or a fragment thereof (e.g., the extracellular domain) can belabeled with a detectable moiety such as ¹²⁵I. Alternatively, anotherdetectable moiety such as biotin, avidin, or an enzyme that can catalyzea colorometric or fluorometric reaction may be used. Cells to be testedfor 4-1BB expression are contacted with the labeled 4-1BB-L then washedto remove unbound labeled 4-1BB-L. Cells that bound the labeled 4-1BB-Lare detected via the detectable moiety. Likewise, the human 4-1BB of thepresent invention is useful as a research reagent in binding assays todetect cells expressing 4-1BB-L. Identifying additional cell typesexpressing 4-1BB or 4-1BB-L provides insight into cell types that mayplay a role in the activation and function of cells of the immunesystem, particularly T-cells.

The 4-1BB ligand proteins disclosed herein also may be employed tomeasure the biological activity of 4-1BB protein in terms of bindingaffinity for 4-1BB-L. To illustrate, 4-1BB-L may be employed in abinding affinity study to measure the biological activity of a 4-1BBprotein that has been stored at different temperatures, or produced indifferent cell types. The biological activity of a 4-1BB protein thuscan be ascertained before it is used in a research study, for example.

4-1BB-L proteins find use as reagents that may be employed by thoseconducting “quality assurance” studies, e.g., to monitor shelf life andstability of 4-1BB protein under different conditions. 4-1BB ligands maybe used in determining whether biological activity is retained aftermodification of a 4-1BB protein (e.g., chemical modification,truncation, mutation, etc.). The binding affinity of the modified 4-1BBprotein for a 4-1BB-L is compared to that of an unmodified 4-1BB proteinto detect any adverse impact of the modifications on biological activityof 4-1BB.

A different use of a 4-1BB ligand is as a reagent in proteinpurification procedures. 4-1BB-L or Fc/4-1BB-L fusion proteins may beattached to a solid support material by conventional techniques and usedto purify 4-1BB by affinity chromatography.

Likewise, human 4-1BB may be employed to measure the biological activityof human 4-1BB-L polypeptides in terms of binding affinity. Human 4-1BBfinds further use in purification of human 4-1BB-L by affinitychromatography.

The present invention provides pharmaceutical compositions comprising aneffective amount of a purified 4-1BB-L or 4-1BB polypeptide and asuitable diluent, excipient, or carrier. Such carriers will be nontoxicto patients at the dosages and concentrations employed. Ordinarily, thepreparation of such compositions entails combining a mammalian 4-1BB-Lpolypeptide or derivative thereof with buffers, antioxidants such asascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins, amino acids, carbohydrates including glucose,sucrose or dextrans, chelating agents such as EDTA, glutathione andother stabilizers and excipients. Neutral buffered saline or salinemixed with conspecific serum albumin are exemplary appropriate diluents.

Such compositions may be used to stimulate the immune system in view ofthe inventive proteins' presence and effect on certain cells associatedwith the immune response. For therapeutic use, the compositions areadministered in a manner and dosage appropriate to the indication andthe size and condition of the patient. Administration may be byinjection, continuous infusion, sustained release from implants, orother suitable mode.

Nucleic Acid Fragments

The present invention further provides fragments of the 4-1BB-L andhuman 4-1BB nucleotide sequences presented herein. Such fragmentsdesirably comprise at least about 14 nucleotides. DNA and RNAcomplements of said fragments are provided herein, along with bothsingle-stranded and double-stranded forms of the DNA.

Among the uses of such nucleic acid fragments is use as a probe. Suchprobes may be employed in cross-species hybridization procedures toisolate 4-1BB-L or 4-1BB DNA from additional mammalian species. As oneexample, a probe corresponding to the extracellular domain of 4-BB-L or4-1BB may be employed. The probes also find use in detecting thepresence of 4-1BB-L or 4-1BB nucleic acids in in vitro assays and insuch procedures as Northern and Southern blots. Cell types expressing4-1BB-L or 4-1BB can be identified. Such procedures are well known, andthe skilled artisan can choose a probe of suitable length, depending onthe particular intended application.

Other useful fragments of the 4-1BB-L or 4-1BB nucleic acids areantisense or sense molecules comprising a single-stranded nucleic acidsequence (either RNA or DNA) capable of binding to target 4-1BB-L or4-1BB mRNA (sense) or DNA (antisense) sequences. In one embodiment, theantisense or sense molecule is a nucleotide sequence corresponding orcomplementary to the coding region of the 4-1BB or 4-1BB-L sequencespresented herein or a fragment thereof or the RNA complement thereof.Such oligonucleotides preferably comprise at least about 14 nucleotides,most preferably from about 17 to about 30 nucleotides. The ability toderive an antisense or a sense oligonucleotide based upon a cDNAsequence for a given protein is described in, for example, Stein andCohen, Cancer Res. 48:2659, 1988 and van der Krol et al., BioTechniques6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block translation(RNA) or transcription (DNA) by one of several means, including enhanceddegradation of the duplexes, premature termination of transcription ortranslation, or by other means. The antisense oligonucleotides thus maybe used to block expression of 4-1BB-L proteins.

Antisense or sense oligonucleotides of the present invention furthercomprise oligonucleotides having modified sugar-phosphodiester backbones(or other sugar linkages, such as those described in WO91/06629) andwherein such sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences. Otherexamples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10448, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or other gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, vectors derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application US90/02656).

Sense or antisense oligonucleotides may also be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. The ligand binding molecule should beconjugated in a manner that does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

The following examples are for the purposes of illustrating certainembodiments of the invention, and are not to be construed as limitingthe scope of the invention as claimed herein.

EXAMPLE 1 Preparation of Murine 4-1BB/Fc Fusion Protein for Use inScreening Clones

This example illustrates construction of an expression vector encoding afusion protein comprising a soluble murine 4-1BB polypeptide fused to anFc region polypeptide derived from a human IgG1 antibody. The fusionprotein is used for detecting clones encoding a 4-1BB ligand. Oneadvantage of employing an Fc-containing fusion protein is the facilepurification made possible by the Fc moiety. Other polypeptides derivedfrom an antibody Fc domain, and which bind with relatively high affinityto protein A- or protein G-containing columns, may be substituted forthe Fc polypeptide employed below.

DNA encoding a portion of the extracellular (ligand binding) domain ofthe murine 4-1BB receptor was isolated by polymerase chain reaction(PCR) using primers based upon the sequence published in Kwon et al. IIand presented herein as SEQ ID NOS:5 and 6. A BD14-20 alloreactivemurine T-cell clone was induced with concanavalin A (Con A), usingstandard techniques (Kwon et al. II). Total RNA was isolated from theinduced cells by the guanadinium thiocyanate method (Mosley et al., Cell59: 335 (1989)). cDNA was prepared by conventional techniques and usedas the template in a conventional PCR procedure (Sarki et al., Science239:487, 1988). The 5′ primer oligonucleotide sequence was:

-   -   GTCACTAGTTCTGTGCAGAACTCCTGTGATAAC (SEQ ID NO:9)

SEQ ID NO:9 comprises a SpeI site (double underline) and a signalcleavage site followed by a sequence (underlined) that corresponds tothe nucleotides encoding the first seven amino acids of the maturemurine 4-1BB protein. The 3′ primer sequence was a 35-meroligonucleotide comprising the sequence:

CACAAGATCTGGGCTCCTCTGGAGTCACAGAAATG (SEQ ID NO:10)

The SEQ ID NO:10 oligonucleotide contains a Bgl 2 restriction site(double underline) and a sequence (underlined) that is complementary tonucleotides 510–528 of SEQ ID NO:5.

The PCR reaction was amplified with 30 cycles. The amplified DNAfragment comprised a sequence encoding a soluble murine 4-1BBpolypeptide comprising amino acids 1 (Val) through 153 (Glu) of SEQ IDNO:5, i.e., a fragment of the extracellular domain terminating ten aminoacids upstream of the transmembrane region. The resulting PCR productswere digested with SpeI and BglII restriction enzymes.

A DNA encoding the Fc region of a human IgG1 antibody (to be fused tothe 4-1BB-encoding sequence) was isolated as follows. SEQ ID NO:14 andSEQ ID NO:15 present the nucleotide and encoded amino acid sequences ofa human IgG1 Fc polypeptide-encoding DNA inserted into the polylinker(multiple cloning site) of a pBluescript®SK cloning vector (StratageneCloning Systems, La Jolla, Calif.). Amino acids 1–13 of SEQ ID NO:15 areencoded by the polylinker segment of the vector, and amino acids 14(Glu) through 245 (Lys) constitute the Fc polypeptide. An Fc-encodingDNA fragment 699 base pairs in length was derived by cleaving therecombinant pBluescript®SK vector with BglII (the recognition site forwhich comprises nucleotides 47–52 of SEQ ID NO:14) and SpeI (whichcleaves in the polylinker downstream of the inserted Fc sequence).

The Fc fragment and the murine 4-1BB extracellular domain fragmentisolated by PCR above were cloned into an SpeI-cleaved Smag 4 vector ina three-way ligation. The Smag 4 vector comprises a murine interleukin-7(IL-7) leader sequence inserted into the mammalian high expressionvector pDC201 (described in Sims et al., Science 241:585, 1988, and inPCT application WO 89/03884), which is capable of replication in E.coli. E. coli cells were transfected with the ligation mixture and thedesired recombinant vector (comprising the Fc-encoding DNA joined to theC-terminus of the 4-1BB-encoding DNA via the BglII sites) was isolatedtherefrom.

The gene fusion encoding the soluble 4-1BB/Fc fusion protein was excisedby digesting the recombinant Smag 4 vector with BamHI. The fragmentencoding the fusion protein was isolated, the ends were filled in usingthe Klenow fragment of DNA polymerase I, and the resulting blunt-endedfragment was ligated into a SalI cleaved (blunt ended) dephosphorylatedHAV-EO vector.

The gene fusion was transferred to the HAV-EO vector (described by Doweret al., J. Immunol. 142:4314; 1989) in order to improve expressionlevels. The HAV-EO vector is a derivative of pDC201 and allows for highlevel expression in CV-1/EBNA cells. The CV-1/EBNA cell line (ATCC10478) was derived by transfecting the African green monkey kidney cellline CV-1 (ATCC CCL-70) with a gene encoding Epstein-Barr virus nuclearantigen-1 (EBNA-1), as described by McMahan et al. (EMBO J. 10:2821,1991). The CV-1/EBNA cells constitutively express EBNA-1 driven from thehuman cytomegalovirus (CMV) intermediate-early enhancer/promoter. TheEBNA-1 gene allows for episomal replication of expression vectors suchas HAV-EO that contain the EBV origin of replication.

The recombinant HAV-EO vector containing the 4-1BB/Fc gene fusion wastransfected into CV-1/EBNA cells using standard techniques. Thetransfected cells transiently expressed and secreted a 4-1BB/Fc fusionprotein into the culture supernatant, which was harvested after one weekof cultivation. The 4-1BB/Fc fusion protein was purified by protein Gaffinity chromatography. More specifically, one liter of culturesupernatant, containing the 4-1BB/Fc fusion protein, was passed over asolid phase protein G column, and the column was washed thoroughly withphosphate-buffered saline (PBS). The adsorbed fusion protein was elutedwith 50 mM glycine buffer, pH 3. Purified fusion protein was brought topH 7 with 2 M Tris buffer, pH 9. Silver-stained SDS gels of the purified4-1BB/Fc fusion protein showed it to be >98% pure.

Purified 4-1BB/Fc fusion protein was radioiodinated with ¹²⁵I using acommercially available solid phase reagent (IODO-GEN, Pierce ChemicalCo., Rockford, Ill.). In this procedure, 5 μg of IODO-GEN were plated atthe bottom of a 10×75 mm glass tube and incubated for twenty minutes at4° C. with 75 μl of 0.1M sodium phosphate, pH 7.4 and 20 μl (2 mCi)Na¹²⁵I. The solution was then transferred to a second glass tubecontaining 5 μg of 4-1BB/Fc in 45 μl PBS (phosphate buffered saline) andthis reaction mixture was incubated for twenty minutes at 4° C. Thereaction mixture was fractionated by gel filtration on a 2 ml bed volumeof Sephadex® G-25 (Sigma), and then equilibrated in RPMI 1640 mediumcontaining 2.5% (v/v) bovine serum albumin (BSA), 0.2% (v/v) sodiumazide and 20 mM Hepes, pH 7.4 binding medium. The final pool of¹²⁵I-4-1BB/Fc was diluted to a working stock solution of 1×10⁻⁷M inbinding medium and stored for up to one month at 4° C. withoutdetectable loss of receptor binding activity.

Approximately 50%–60% label incorporation was observed. Radioiodinationyielded specific activities in the range of 1×10¹⁵ to 5×10¹⁵ cpm/nmole(0.42–2.0 atoms of radioactive iodine per molecule of protein). SDSpolyacrylamide gel electrophoresis (SDS-PAGE) revealed a single labeledpolypeptide consistent with expected values. The labeled fusion proteinwas greater than 98% trichloroacetic acid (TCA) perceptible, indicatingthat the ¹²⁵I was covalently bound to the protein.

EXAMPLE 2 Isolation of cDNA Encoding Human 4-1BB

A human cDNA library was screened with a murine 4-1BB DNA probe in aneffort to isolate cDNA encoding a human 4-1BB by cross-specieshybridization. The degree of homology between murine 4-1BB and human4-1BB DNA was not known prior to isolation and sequencing of human 4-1BBDNA by the following procedure.

A fragment of the murine 4-1BB DNA of SEQ ID NO:5 was isolated bypolymerase chain reaction (PCR) using conventional procedures. Thetemplate was cDNA synthesized using a first strand cDNA synthesis kit(Stratagene Cloning Systems, La Jolla, Calif.) on RNA isolated from theinduced murine T cell clone BD14-20 (see example 1). The 5′ primer wasthe oligonucleotide presented as SEQ ID NO:9 and described in example 1.The 3′ primer was the following oligonucleotide:

5′ CAGACTAGTTCACTCTGGAGTCACAGAAATG 3′ (SEQ ID NO:11)

This oligonucleotide comprises an SpeI site (double underline) and asequence (underlined) that is complementary to nucleotides 510–528 ofSEQ ID NO:5 (murine 4-1BB). The amplified PCR products (comprisingnucleotides 70–528 of the murine 4-1BB sequence of SEQ ID NO:5) wereligated into a SmaI-digested pBLUESCRIPT®SK cloning vector (StratageneCloning Systems, La Jolla, Calif.). E. coli cells were transfected withthe ligation mixture, and the desired recombinant vector was recovered.The murine 4-1BB DNA insert was excised by digesting the recombinantvector with NotI and EcoRI. The excised DNA was labeled with ³²P using aconventional random priming technique.

The labeled murine 4-1BB DNA fragment was used to screen a human cDNAlibrary that was constructed as described by Park et al. (Blood 74:56,1989). Briefly, the cDNA library was derived from poly A⁺ RNA isolatedfrom human peripheral blood T-lymphocytes (purified by E rosetting) thathad been activated for 18 hours with phytohemagglutinin (PHA) andphorbol myristate acetate (PMA). Blunt-ended cDNA was methylated andEcoRI linkers were attached, followed by ligation to λgt10 arms andpackaging into phage λ extracts (Stratagene Cloning Systems, La Jolla,Calif.) according to the manufacturer's instructions.

Hybridization was conducted at 37° C. in 50% formamide, followed bywashing in 2×SSC, 0.1% SDS at 55° C. The cDNA insert of a hybridizingclone was isolated and sequenced. The nucleotide and encoded amino acidsequences of this human 4-1BB cDNA are presented in SEQ ID NO:7 and SEQID NO:8.

The human 4-1BB protein comprises an N-terminal signal peptide (aminoacids −23 to −1 of SEQ ID NO:8), an extracellular domain comprisingamino acids 1–163, a transmembrane region comprising amino acids164–190, and a cytoplasmic domain comprising amino acids 191–232. Thehuman 4-1BB of SEQ ID NO:7 is 60% identical to murine 4-1BB at the aminoacid level, and 71% identical at the DNA level.

EXAMPLE 3 Preparation of Human 4-1BB/Fc Fusion Protein for Use inScreening Clones

This example illustrates construction of an expression vector encoding afusion protein comprising the extracellular domain of human 4-1BB fusedto the N-terminus of an Fc region polypeptide derived from a human IgG1antibody. The fusion protein was used for detecting clones encoding ahuman 4-1BB ligand.

A DNA fragment encoding a soluble human 4-1BB was isolated by PCR usingthe human 4-1BB cDNA synthesized in example 2 as a template. The 5′primer was the following oligonucleotide:

5′ ATAGCGGCCGC TGCCAGATTTCATCATGGGAAAC 3′ (SEQ ID NO:12)

This oligonucleotide comprises a NotI site (double underlined) and asegment (underlined) corresponding to nucleotides 106–128 of SEQ IDNO:7.

The 3′ primer was the following oligonucleotide:

The oligonucleotide comprises a Bgl II site (double underlined) and asegment (underlined) complementary to nucleotides 653–677 of SEQ IDNO:7. The segment with a dotted underline is complementary tonucleotides 41–46 of SEQ ID NO:14 and serves to replace the codons forthe first two amino acids of the Fc polypeptide (amino acids 14 and 15of SEQ ID NO:14), which are upstream of the BglII site.

A DNA fragment encoding an antibody Fc region polypeptide was isolatedby cleaving a recombinant vector comprising Fc-encoding DNA inpBLUESCRIPT®SK (described in example 1) with BglII and NotI. BglIIcleaves near the 5′ end of the Fc DNA, as described in example 1, andNotI cleaves in the polylinker of the vector downstream of the insertedFc-encoding DNA.

In a 3-way ligation, the soluble human 4-1BB polypeptide-encoding DNAisolated by PCR above and the Fc-encoding BglII/NotI fragment wereligated into a NotI-digested expression vector pDC406 (described inMcMahan et al., EMBO J., 10:2821, 1991). E. coli cells were transformedwith the ligation mixture and the desired recombinant vector wasrecovered. The fusion protein encoded by this vector comprised aminoacids −23 to 163 of SEQ ID NO:8 (a soluble human 4-1BB polypeptideconsisting of the signal peptide and the entire extracellular domain)followed by amino acids 14–245 of SEQ ID NO:15 (Fc polypeptide).CV1-EBNA cells (described in example 1) were transfected with therecombinant vector and cultured to produce and secrete the soluble human4-1BB/Fc fusion protein. The fusion protein was purified by protein Gaffinity chromatography for use in identifying clones expressing human4-1BB ligand, as described in example 5 below.

EXAMPLE 4 Isolation of Murine 4-1BB Ligand cDNA

This example describes the isolation of cDNA encoding a murine 4-1BBligand (4-1BB-L) using a direct expression cloning technique. Theprocedure was as follows.

Several cell lines were screened for the ability to bind theradioiodinated murine 4-1BB/Fc fusion protein described in Example 1.Briefly, quantitative binding studies were performed according tostandard methodology, and Scatchard plots were derived for the variouscell lines. A clonal cell line designated EL4 6.1C10 was identified asexpressing approximately 1500 molecules of a 4-1BB/Fc-binding proteinper cell, with an affinity binding constant of approximately 2×10⁹ M⁻¹.The EL4 6.1C10 cell line was derived from a subclone designated EL4 6.1by using a cell sorter to enrich for a cell population expressing highlevels of murine type I Interleukin-1 receptor, as described in U.S.Pat. No. 4,968,607. EL4 6.1 had been derived from a mouse thymoma cellline EL-4 (ATCC TIB 39) as described by MacDonald et al. (J. Immunol.135:3944, 1985) and Lowenthal and MacDonald (J. Exp. Med. 164:1060,1986).

A cDNA library was derived from the EL4 6.1C10 cell line using a libraryconstruction technique substantially similar to that described byAusubel et al., eds., Current Protocols in Molecular Biology, Vol. 1,(1987). Total RNA was extracted from the EL4 6.1C10 cell line, poly (A)⁺mRNA was isolated by oligo dT cellulose chromatography, anddouble-stranded cDNA was made substantially as described by Gubler etal., Gene 25:263 (1983). Briefly, poly(A)⁺ mRNA fragments were convertedto RNA-cDNA hybrids by reverse transcriptase using randomhexanucleotides as primers. The RNA-cDNA hybrids were then convertedinto double-stranded cDNA fragments using RNAse H in combination withDNA polymerase I. The resulting double-stranded cDNA was blunt-endedwith T4 DNA polymerase, ligated into SmaI-cleaved, dephosphorylatedexpression vector pDC201 (described in Sims et al., Science 241:585,1988, and in PCT application WO 89/03884), and transformed intocompetent E. coli DH5α cells.

Plasmid DNA was isolated from pools consisting of approximately 2,000clones of transformed E. Coli per pool. The isolated plasmid DNA wastransfected into a sub-confluent layer of COS cells using DEAE-dextranfollowed by chloroquine treatment substantially according to theprocedures described in Luthman et al. (Nucl. Acids Res. 11:1295, 1983)and McCutchan et al. (J. Natl. Cancer Inst. 41:351, 1986). Briefly, COScells were maintained in complete medium (Dulbecco's modified Eagles'media containing 10% (v/v) fetal calf serum, 50 U/ml penicillin, 50 U/mlstreptomycin, and 2 mM L-glutamine and were plated to a density ofapproximately 2×10⁵ cells/well in single-well chambered slides(Lab-Tek). The slides were pre-treated with 1 ml human fibronectin (10μg/ml PBS) for 30 minutes followed by a single washing with PBS. Mediawas removed from the monolayer of adherent cells and replaced with 1.5ml complete medium containing 66.6 μM chloroquine sulfate. About 0.2 mlof a DNA solution (2 μg DNA, 0.5 mg/ml DEAE-dextran in complete mediumcontaining chloroquine) was added to the cells and the mixture wasincubated at 37° C. for about five hours. Following incubation, mediawas removed and the cells were shocked by addition of complete mediumcontaining 10% DMSO (dimethylsulfoxide) for 2.5–20 minutes. Shocking wasfollowed by replacement of the solution with fresh complete medium. Thecells were grown in culture to permit transient expression of theinserted DNA sequences. These conditions led to an 80% transfectionfrequency in surviving COS cells.

After 48–72 hours in culture, monolayers of transfected COS cells wereassayed by slide autoradiography for expression of a protein that bindsthe radioiodinated murine 4-1BB/Fc fusion protein prepared in Example 1.The slide autoradiography technique was essentially as described byGearing et al. (EMBO J., 8:3667, 1989). Briefly, the transfected COScells were washed once with binding medium (RPMI 1640 containing 25mg/ml bovine serum albumin (BSA), 2 mg/ml sodium azide, 20 mM HEPES pH7.2, and 50 mg/ml nonfat dry milk) and incubated for 2 hours at 4° C. inbinding medium containing 1×10⁻⁹ M ¹²⁵I-4-1BB/Fc fusion protein. Afterincubation, cells in the chambered slides were washed three times withbinding medium, followed by two washes with PBS, (pH 7.3) to removeunbound radiolabeled fusion protein.

The cells were fixed by incubating in 10% glutaraldehyde in PBS (30minutes at room temperature), washed twice in PBS and air-dried. Theslides were dipped in Kodak GTNB-2 photographic emulsion (6× dilution inwater) and exposed in the dark for four days at room temperature in alight-proof box. The slides were developed in Kodak D19 developer,rinsed in water and fixed in Agfa G433C fixer. The slides wereindividually examined under a microscope at 25–40× magnification.Positive slides showing cells expressing 4-1BB ligand were identified bythe presence of autoradiographic silver grains against a lightbackground.

One pool containing approximately 2120 individual clones was identifiedas potentially positive for binding the 4-1BB/Fc fusion protein. Thepool was broken down into smaller pools of approximately 250 colonies,from which DNA was isolated and transfected into COS-7 cells. Thetransfectants were screened by slide autoradiography as described above.Three positive pools were identified. Plasmid DNA isolated fromindividual colonies corresponding to the three positive pools wastransfected into COS cells and screened by the same procedure.

A single clone encoding a protein that binds murine 4-1BB/Fc wasisolated. Plasmid DNA was isolated from the clone, and the nucleotidesequence of the cDNA insert in the recombinant vector was determined.The cloned cDNA was found to encode a novel protein, a murine 4-1BBligand (4-1BB-L) protein of the present invention. The nucleotidesequence of the isolated murine 4-1BB-L cDNA and the amino acid sequenceencoded thereby are presented in SEQ ID NO:1 and SEQ ID NO:2. E. coliDH5α cells transformed with a recombinant vector comprising the murine4-1BB-L-encoding cDNA of SEQ ID NO:1 in the mammalian expression vectorpDC201 were deposited with the American Type Culture Collection,Rockville, Md., on Sep. 5, 1991, under accession no. ATCC 68682. Themurine 4-1BB-L is a type II protein comprising a cytoplasmic domain(amino acids 1–82 of SEQ ID NO:1); a transmembrane region (amino acids83–103 of SEQ ID NO:1); and an extracellular domain (amino acids 104–309of SEQ ID NO:1).

EXAMPLE 5 Isolation of cDNA Encoding a Human 4-1BB Ligand

Different cell lines were screened by flow cytometry for the ability tobind the human 4-1BB/Fc (hu 4-1BB/Fc) fusion protein prepared in Example3. Cells were initially incubated with hu4-1BB/Fc (10 μg/ml), followedby biotinylated goat anti-human IgG, Fc-specific (Jackson ImmunoResearchLaboratories, West Grove, Pa.) and finally streptavidin-phycoerythrin(Becton-Dickinson). Flow cytometry was performed using a FACScan(Becton-Dickinson) and data were collected on 104 viable cells. Analloreactive CD4⁺ human T cell clone designated PL1 stimulated with ananti-CD3 antibody exhibited 4-1BB/Fc binding. The receptor binding wasdetectable 30 minutes after stimulation and peaked 2–4 hourspost-stimulation.

Since peak production of mRNA would precede peak production of the4-1BB-binding protein translated therefrom, total RNA was isolated fromthe PL1 cells 90 minutes after stimulation, and poly(A⁺) RNA wasisolated by oligo(dT) cellulose chromatography. cDNA was synthesized onthe poly(A)⁺ RNA template using oligo(dT) primers and a cDNA synthesiskit (Pharmacia Biotech, Inc., Piscataway, N.J.). The resultingdouble-stranded cDNA was ligated into the BglII site of the mammalianexpression vector pDC410 by a BglII adaptor method similar to thatdescribed by Haymerle et al. (Nucl. Acids Res. 14:8615, 1986).

The pDC410 vector is similar to pDC406 (McMahan et al., EMBO J.,10:2821, 1991). In pDC410, the EBV origin of replication of pDC406 isreplaced by DNA encoding the SV40 large T antigen (driven from an SV40promoter). The pDC410 multiple cloning site (mcs) differs from that ofpDC406 in that it contains additional restriction sites and three stopcodons (one in each reading frame). A T7 polymerase promoter downstreamof the mcs facilitates sequencing of DNA inserted into the mcs. E. colistrain DH5α cells were transfected with the cDNA library in pDC410.

Plasmid DNA was isolated from the transformed E. coli cells, pooled,(each pool consisting of plasmid DNA from approximately 1000 individualcolonies) and transfected into a sub-confluent layer of CV-1 EBNA cells(described in example 1). The transfection procedure was theDEAE-dextran followed by chloroquine treatment technique essentially asdescribed in Luthman et al., Nucl. Acids Res. 11:1295 (1983), McCutchanet al., J. Natl. Cancer Inst. 41:351 (1986). Prior to transfection, theCV1-EBNA cells were plated in single-well chambered slides (Lab-Tek) andgrown in culture for two to three days to permit transient expression ofthe inserted DNA sequences.

The transfected cells then were assayed by slide autoradiography forexpression of 4-1BB-L. The assay procedure was similar to that describedin example 4, except that the transfected cells were incubated with tworeagents. The cells were first washed with binding medium containingnonfat dry milk (BM-NFDM) and incubated with the human 4-1BB/Fc fusionprotein prepared in Example 3, in non-radiolabeled form (1 μg/ml inBM-NFDM) for one hour at room temperature. After washing three timeswith BM-NFDM, cells were incubated with 40 ng/ml ¹²⁵I-mouse anti-humanFc antibody (a 1:50 dilution) for one hour at room temperature. Themouse anti-human Fc antibody was obtained from Jackson ImmunoresearchLaboratories, Inc, West Grove, Pa., and radiolabeled by the chloramine Tmethod. After washing three times with BM-NFDM and twice with PBS, cellwere fixed in glutaraldehyde and slides were processed as described inexample 4.

The pool appearing to be most strongly positive was broken down intosmaller pools. DNA from the smaller pools was transfected into CV1-EBNAcells and screened by slide autoradiography as described above. Positivepools were identified, and DNA from individual colonies corresponding tothe positive pools was screened by the foregoing procedure. Twoindividual clones expressing 4-1BB-L proteins were isolated. Thenucleotide sequence of the human 4-1BB-L cDNA insert of one of theclones (clone 7A) and the amino acid sequence encoded thereby ispresented in SEQ ID NO:3 and SEQ ID NO:4. This human 4-1BB-L proteincomprises a cytoplasmic domain (amino acids 1–25 of SEQ ID NO:4), atransmembrane region (amino acids 26–46), and an extracellular domain(amino acids 47–252).

The human 4-1BB-L amino acid sequence of SEQ ID NO:3 is about 33%identical to the murine 4-1BB-L amino acid sequence of SEQ ID NO:1, andthe nucleotide sequences are about 50% identical. The recombinant vectorof clone 7A, i.e., human 4-1BB-L cDNA in vector pDC410, designatedhu4-1BB-L (7A)/pDC410, was deposited in E. coli DH5α with the AmericanType Culture Collection, Rockville, Md. on Apr. 16, 1993, underaccession number ATCC 69285.

EXAMPLE 6 Expression of Biologically Active Soluble 4-1BB-L in MammalianCells

Soluble 4-1BB-L was expressed in a monkey kidney cell line designatedCV-1 (ATCC CCL 70). The expressed protein was biologically active inthat it bound a 4-1BB/Fc fusion protein.

The soluble 4-1BB-L was produced as follows. DNA encoding amino acids106 through 309 of the murine 4-1BB-L of SEQ ID NO:1 was isolated by PCRusing oligonucleotide primers based on the nucleotide sequence presentedin SEQ ID NO:1. The amplified DNA was inserted into the mammalianexpression vector designated HAV-EO (described in example 1). cDNAencoding a heterologous (murine interleukin-7) leader peptide, describedin U.S. Pat. No. 4,965,195 which is hereby incorporated by reference,was fused to the N-terminus of the 4-1BB-L cDNA to promote secretion ofthe soluble 4-1BB-L from the host cells. CV-1 cells were transformedwith the resulting recombinant expression vector by conventionaltechniques.

The transformed CV-1 cells were cultured to permit expression andsecretion of the soluble 4-1BB-L into the supernatant. Variousconcentrations of the supernatant were tested in a competitive bindingassay for the ability to inhibit binding of soluble murine 4-1BB/Fc toEL4 6.1C10 cells. The soluble murine 4-1BB/Fc fusion protein wasproduced as described in example 1. The murine EL4 6.1C10 cell lineexpresses cell surface 4-1BB-L, as described in example 4.

The results of the assay, presented in FIG. 1, demonstrate that thesoluble murine 4-1BB-L protein expressed in CV-1 cells inhibits bindingof a soluble murine 4-1BB/Fc fusion protein to EL4 6.1C10 cells. Thesoluble 4-1BB-L protein's ability to inhibit binding of 4-1BB/Fc to thecells indicates that the soluble 4-1BB-L is binding to the 4-1BB/Fc.

An expression vector encoding soluble human 4-1BB-L can be substitutedfor the murine 4-1BB-L-encoding vector in the foregoing procedure.Likewise, human 4-1BB/Fc would be substituted for murine 4-1BB/Fc, andhuman cells expressing 4-1BB employed in place of murine cells, in thecompetitive binding assay.

FIG. 2 presents the result of a control experiment in which CV-1 cellswere transformed with an “empty” HAV-EO vector (lacking any inserted4-1BB-L DNA). Supernatant from a culture of the transformed cells didnot inhibit binding of 4-1BB/Fc to EL4 6.1C10 cells when tested in thecompetitive binding assay.

EXAMPLE 7 Expression of Biologically Active Soluble 4-1BB-L in Yeast

Soluble recombinant 4-1BB-L expressed in yeast cells (Saccharomycescerevisiae) was shown to be biologically active in that the expressedprotein was able to bind a 4-1BB/Fc fusion protein. The 4-1BB-L proteinwas produced by inserting cDNA encoding amino acids 106 through 309 ofthe murine 4-1BB-L of SEQ ID NO:1 (isolated and amplified by PCR) intoan expression vector comprising an ADH2 promoter (described above). Theexpression vector also contained DNA encoding the yeast α-factor leaderpeptide (described above) fused to the 5′ end of DNA encoding a FLAG®peptide, which was fused to the 5′ end of the 4-1BB-L DNA. The FLAG®octapeptide constitutes an epitope reversibly bound by a particularmonoclonal antibody, which facilitates purification of recombinantproteins (4-1BB-L in this case), as described in U.S. Pat. No.5,011,912. The octapeptide may be removed using bovine mucosalenterokinase, which specifically cleaves at the residue immediatelyfollowing the DK pairing.

S. cerevisiae cells were transformed with the resulting recombinantexpression vector by conventional techniques. The transformed cells werecultured to permit expression and secretion of the soluble 4-1BB-L intothe supernatant. Various concentrations of the supernatant were testedin a competitive binding assay for the ability to inhibit binding ofsoluble murine 4-1BB/Fc to EL4 6.1C10 cells. The soluble murine4-1BB-/Fc fusion protein was produced as described in example 1. MurineEL4 6.1C10 cells express cell surface 4-1BB-L, as described in example4.

FIG. 3 presents the results of the competitive binding assay, whichdemonstrates that the soluble murine 4-1BB-L protein expressed in S.cerevisiae cells inhibits binding of the murine 4-1BB/Fc fusion proteinto EL4 6.1C10 cells. The 4-1BB-L protein thus is able to bind 4-1BB/Fc.Although recombinant 4-1BB-L can be expressed in yeast cells, mammaliancells are preferred as host cells. The specific activity of 4-1BB-Lproduced in yeast generally is lower than that of 4-1BB-L produced inmammalian cells such as CV-1.

EXAMPLE 8 Monoclonal Antibodies that Bind 4-1BB-L or 4-1BB

Murine 4-1BB-L or human 4-1BB-L protein may be purified by 4-1BB/Fcaffinity chromatography as described above. Full length 4-1BB-L orimmunogenic fragments thereof (e.g., the extracellular domain) can beused as an immunogen to generate monoclonal antibodies usingconventional techniques, for example, those techniques described in U.S.Pat. No. 4,411,993. Another alternative involves using a soluble4-1BB-L/Fc fusion protein, comprising the extracellular domain of a4-1BB-L fused to an antibody Fc polypeptide, as the immunogen.

Briefly, mice are immunized with 4-1BB-L as an immunogen emulsified incomplete Freund's adjuvant, and injected subcutaneously orintraperitoneally in amounts ranging from 10–100 μg. Ten to twelve dayslater, the immunized animals are boosted with additional 4-1BB-Lemulsified in incomplete Freund's adjuvant. Mice are periodicallyboosted thereafter on a weekly to bi-weekly immunization schedule. Serumsamples are periodically taken by retro-orbital bleeding or tail-tipexcision for testing by dot blot assay or ELISA (Enzyme-LinkedImmunosorbent Assay), for antibodies that bind 4-1BB-L.

Following detection of an appropriate antibody titer, positive animalsare provided one last intravenous injection of 4-1BB-L in saline. Threeto four days later, the animals are sacrificed, spleen cells harvested,and spleen cells are fused to a murine myeloma cell line (e.g., NS1 orAg 8.653). The latter myeloma cell line is available from the AmericanType Culture Collection as P3x63Ag8.653 (ATCC CRL 1580). Fusionsgenerate hybridoma cells, which are plated in microtiter plates in a HAT(hypoxanthine, aminopterin and thymidine) selective medium to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

The hybridoma cells are screened by ELISA for reactivity againstpurified 4-1BB-L by adaptations of the techniques disclosed in Engvallet al., Immunochem. 8:871 (1971) and in U.S. Pat. No. 4,703,004. Apreferred screening technique is the antibody capture techniquedescribed in Beckmann et al., (J. Immunol. 144:4212, 1990). Positivehybridoma cells can be injected intraperitoneally into syngeneic BALB/cmice to produce ascites containing high concentrations of anti-4-1BB-Lmonoclonal antibodies. Alternatively, hybridoma cells can be grown invitro in flasks or roller bottles by various techniques. Monoclonalantibodies produced in mouse ascites can be purified by ammonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to protein A orprotein G can be used, as can affinity chromatography based upon bindingto 4-1BB-L.

Purified human 4-1BB or immunogenic fragments thereof (e.g., a fragmentderived from the extracellular domain) may be substituted for the4-1BB-L immunogens in the foregoing procedures. In one embodiment, asoluble human 4-1BB/Fc fusion protein (e.g., as described in example 3)is employed as the immunogen. Monoclonal antibodies that bind human4-1BB thus are prepared.

EXAMPLE 9 Dimeric Forms of the Inventive Proteins

Preparation of fusion proteins comprising an antibody Fc polypeptidefused to the C-terminus of a soluble human 4-1BB polypeptide (suchfusion proteins being referred to as 4-1BB/Fc hereinafter) is describedin example 3. Disulfide bonds form between the Fc moieties, as inantibodies, resulting in dimers comprising two 4-1BB/Fc polypeptides.Such dimers may be recovered from cultures of cells expressing the4-1BB/Fc fusion proteins.

Dimers comprising two 4-1BB-L/Fc polypeptides joined via disulfide bondsmay be prepared by analogous procedures. A DNA fragment encoding asoluble 4-1BB-L polypeptide is isolated by procedures described above(e.g., using oligonucleotides that define the desired termini of thefragment as primers in PCR). An expression vector comprising theisolated fragment fused to an Fc-encoding fragment is constructed byprocedures analogous to those described in examples 1 and 3. The Fcpolypeptide is preferably fused to the N-terminus of the 4-1BB-Lpolypeptide, however. Dimers of 4-1BB-L/Fc are recovered from culturesof cells transformed with the expression vector.

The dimers preferably are produced in 293 cells (ATCC CRL 1573). The 293cell line was derived from transformed primary human embryonal kidneycells.

EXAMPLE 10 Cross-Species Reactivity

Inhibition studies were used to investigate cross-species binding of4-1BB to its ligand. 2.5×10⁶ EL4 6.1 cells (murine thymoma subclone)expressing 1800 mu4-1 BB surface ligands/cell were incubated with 0.1 nM¹²⁵I-mu4-1BB/Fc (1×10¹⁵ cpm/mmole) and serially diluted, unlabeled humanor murine 4-1BB/Fc in a total volume of 150 μl binding media for 2 hoursat 4° C. Duplicate aliquots were microfuged through a phthalate oilmixture in 400 μl plastic tubes (essentially as described in Smith etal., Cell 73:1349, 1993) to separate bound and free 4-1BB/Fc. The tubeswere cut, and top (free) and bottom (bound) 4-1BB/Fc counted.Nonspecific binding was determined by inclusion of a 200-fold molarexcess of unlabeled mu4-1BB/Fc.

Unlabeled mu4-1BB/Fc completely inhibited ¹²⁵I-mu4-1BB/Fc binding tonative surface mu4-1BB-L. Unlabeled hu4-1BB/Fc, however, showed nodetectable competition with ¹²⁵I-mu4-1BB/Fc for binding to native murineligand.

Cross-species binding was also assessed qualitatively with the sensitiveslide autoradiography assay. Consistent with the inhibition studies,hu4-1BB/Fc did not bind recombinant mu4-1BB-L expressed on the surfaceof CV-1 cells, and no binding of mu4-1BB/Fc to CV-1 cells expressinghu4-1BB-L was detected. Thus, there appears to be no significantligand/receptor cross-reactivity between human and mouse species.

EXAMPLE 11 Expression of Human 4-1BB-L mRNA

Northern blot analysis demonstrated the presence of multiple sizeclasses of hu4-1BB-L mRNA transcripts. 4-1BB-L message was absent inresting PL-1 cells, but was present within 30 minutes after stimulationwith immobilized anti-CD3 mAb, peaking at approximately one hour afterstimulation. A hybridoma designated OKT3 that produces an anti-CD3monoclonal antibody is available from ATCC under the designation CRL8001.

Transcripts were also observed in a variety of other human cell linessuch as the EBV-transformed human B cell line MP-1, the monocytic cellline THP-1, the Mo-7E megakaryocytic cell line and the neuroblastomaSK-N-SH. Human 4-1BB-L transcripts were absent in RNA isolated from theAML cell line KG-1. A Northern blot of RNAs from various human tissues(Clonetech, Palo Alto, Calif.) was also probed, which demonstrated theexpression of 4-1BB-L transcripts in brain, placenta, lung, skeletalmuscle and kidney. Transcripts were either not present, or present invery low amounts in heart, liver and pancreas.

EXAMPLE 12 Expression of Endogenous Human 4-1BB

A monoclonal antibody reactive with human 4-1BB was generated using thesoluble human 4-1BB/Fc fusion protein of example 3 to immunize BALB/cJmice, and screening for reactivity with hu4-1BB/Fc but not human IgG1 byELISA. This monoclonal antibody (IgG1 isotype) was employed to analyzethe expression of hu4-1BB on a variety of primary human cells and celllines. Northern blot analysis was also conducted. Human 4-1BB protein ormessage (detected by the antibody or the blot, respectively) wasdetected for activated primary T-cells, the alloreactive CD4⁺ T-cellclone PL-1, EBV transformed B cell lines, the pro-monocytic cell lineU937, and resting and activated peripheral blood monocytes.

EXAMPLE 13 Effect of 4-1BB-L on T-Cell Proliferation

(a) Peripheral Blood T-Cells

The ability of human 4-1BB-L to costimulate T-cell proliferation wasassessed in a 3 day tritiated thymidine-incorporation assay. The assayprocedure was generally as described by Goodwin et al. (Cell 73:447,1993). Briefly, human peripheral blood T-cells were isolated andcultured with a titration of fixed CV-1/EBNA cells transfected witheither empty vector or an expression vector containing DNA encoding fulllength hu4-1BB-L, in the presence of suboptimal PHA (0.1%) as acostimulus. After 3 days, cultures were pulsed with [³H] thymidine andincorporated radioactivity was assessed 6 hours later.

The results are shown in FIG. 4. The open circles represent theCV-1/EBNA cells transfected with the empty expression vector, and closedcircles represent the CV-1/EBNA cells transfected with thehu4-1BB-L-encoding expression vector.

The CV-1/EBNA cells expressing recombinant hu4-1BB-L markedly enhancedT-cell proliferation induced by sub-optimal PHA, whereas controlCV-1/EBNA cells had no effect. The hu4-1BB-L had no effect on T-cellproliferation in the absence of a costimulus.

Another thymidine incorporation assay was conducted as described above,except that 10⁴ CV-1/EBNA cells were employed, rather than a titration.Additional controls included soluble hu4-1BB/Fc plus the cellsexpressing hu4-1BB-L; and a soluble human p80 tumor necrosis factorreceptor (TNF-R)/Fc fusion protein plus the cells expressing hu4-1BB-L.Enhanced T-cell proliferation was again observed for T-cells culturedwith PHA and cells expressing hu4-1BB-L. This enhancement of T-cellproliferation was specifically blocked by hu4-1BB/Fc but not byTNF-R/Fc.

(b) T-Cell Clone

The effect of hu4-1BB-L on a long term cultured T-cell clone was alsoanalyzed. Chronically activated T-cells, such as long-term grown T-cellclones (TCC), are induced to undergo programmed cell death whenstimulated with mitogens such as anti-CD3 mAb or PHA in the absence ofantigen-presenting cells (Wesselborg et al., J. Immunol. 150:4338,1993). Since TCC express 4-1BB, we assessed the effect of 4-1BB-L on thegrowth of the alloreactive CD4⁺ human T-cell clone designated PL-1.

PL-1 cells were cultured for 3 days in the presence or absence ofsuboptimal PHA (0.1%) as costimulus and CV-1 cells transfected witheither empty vector (control) or the expression vector containing DNAencoding full length human 4-1BB-L. Viability was determined by trypanblue exclusion.

4-1BB-L had no effect on PL-1 viability or growth in the absence of acostimulus. However, in the presence of PHA, addition of CV-1/EBNA cellsexpressing 4-1BB-L reduced the viability of PL-1 cells from 57% to 31%.4-1BB-L thus enhanced activation-induced cytolysis of the PL-1 cells.

1. A purified polypeptide comprising amino acids 1–163 of SEQ ID NO:8.2. A purified polypeptide comprising amino acids 1–232 of SEQ ID NO:8.3. An isolated DNA comprising a polynucleotide selected from the groupconsisting of: a) nucleotides 120–884 of SEQ ID NO:7; b) nucleotides189–884 of SEQ ID NO:7; and c) a polynucleotide that is degenerate as aresult of the genetic code to a nucleotide sequence of (a) or (b).
 4. Avector comprising a DNA according to claim
 3. 5. A process for preparinga 4–1BB polypeptide, comprising culturing a host cell comprising avector according to claim 4 under conditions that promote expression ofthe 4–1BB polypeptide.
 6. An isolated polynucleic acid comprising atleast about 30 nucleotides of a DNA according to claim 3 or its DNA orRNA complement.
 7. An isolated DNA encoding a polypeptide selected fromthe group consisting of polypeptides comprising amino acids 1–163 of SEQID NO:8 and polypeptides comprising a fragment of amino acids 1–163 ofSEQ ID NO:8, the fragment being capable of binding a 4-1BB-L.
 8. Anisolated DNA of claim 7, wherein said DNA additionally encodes anantibody Fc polypeptide fused to the C-terminus of said polypeptide. 9.A vector comprising a DNA according to claim
 8. 10. A process forpreparing a fusion protein comprising an antibody Fc polypeptide fusedto the C-terminus of a soluble 4–1BB polypeptide, comprising culturing ahost cell comprising a vector according to claim 9 under conditions thatpromote expression of the fusion protein.
 11. A vector comprising a DNAaccording to claim
 7. 12. A process for preparing a soluble 4–1BBpolypeptide, comprising culturing a host cell comprising a vectoraccording to claim 11 under conditions that promote expression of thesoluble 4–1BB polypeptide.
 13. A purified polypeptide comprising theN-terminal amino acid sequenceLeu-Gln-Asp-Pro-Cys-Ser-Asn-Cys-Pro-Ala-Gly-Thr- (amino acid residues1–12 of SEQ ID NO:8), the polypeptide being capable of binding 4-1BB-L.14. A purified polypeptide, comprising an amino acid sequence selectedfrom the group consisting of amino acids 1–232 of SEQ ID NO:8 and aminoacids 1–163 of SEQ ID NO:8.
 15. A purified polypeptide, comprising anamino acid sequence that is identical to a sequence selected from thegroup consisting of amino acids 1–232 of SEQ ID NO:8 and amino acids1–163 of SEQ ID NO:8, except for conservative amino acidsubstitution(s).
 16. A purified polypeptide selected from the groupconsisting of polypeptides comprising amino acids 1–163 of SEQ ID NO:8and polypeptides comprising a fragment of amino acids 1–163 of SEQ IDNO:8, the fragment being capable of binding a 4-1BB-L.
 17. A purifiedpolypeptide of claim 16, additionally comprising an antibody Fcpolypeptide fused to the C-terminus of said polypeptide.
 18. A dimercomprising two polypeptides of claim 17, joined via disulfide bondsbetween the Fc polypeptides fused to said polypeptides.
 19. Acomposition comprising a polypeptide of claim 16 in admixture with adiluent, carrier, or excipient.